The ability of the NMDA receptor antagonistmemantine HCl to provide prophylacticanti-migraine properties in Danio rerio

LTP MIGRAINE THEORY

In situ hybridization

METHODS

RESULTS

(BM-purple)

(anti-DIG Antibody)

(VE-cadherin)

(DIG attached to Uracil)

1. Target mRNA sequence determined

2. Tissue specific RNA probe synthesized

3. Embryos rehydrated

4. Proteinase K used to “poke holes” in the embryo for the RNA probe

5. RNA probe (VE-cadherin) allowed to bind to target mRNA

6. Anti-DIG antibody used to bind to the probe

7. Embryos undergo several wash stages to rid non-specific binding

8. BM-purple substrate applied to show expression in areas of probe binding (specific to blood vessels)

9. Embryos monitored to stop color reaction at the correct time

10. Embryos fixed in 4% PFA to “anchor” the staining

1 2

3 4

5 6

Nitric Oxide Uptake• Endothelium of blood

vessels become dilated because of Nitric Oxide receptor intake of Nitric Oxide itself into smooth muscle of vessels.

• This dilation causes an increase in intracranial pressure.

• This pressure increase is the cause of migraine pain.

Particular interest in off-label uses of currently approved medications

Migraines are classified as a disease and are suffered by over 32 million Americans, roughly 10% of the population.  Study by Dr. Krusz showed promise in the use of memantine HCl as prophylactic treatment for migraine. Migraine occurrence fell 56% in these patients, but the concept warranted further investigation due to its questionable validity (omission of placebo/small sample size).  Took the concept into zebrafish embryos (Danio rerio), which are excellent animal models for disease study due to their transparency and similarity to humans in neurological anatomy and physiology.  In the vascular theory of migraine, blood vessels become dilated, increasing intracranial pressure and causing pain.  Namenda (memantine HCl) only approved for use in the treatment of Alzheimer’s Disease but could prove useful for other conditions and/or diseases, such as Migraines.

In situ hybridization, a useful immunohistochemical staining technique, was used to visualize the blood vessels in the transparent embryos.

Weak electrical stimulation of the presynaptic neuron causes the release of glutamate from the

axon terminal. This glutamate binds to both AMPA and NMDA receptors. Although both

receptors are permeable to Na+ and K+ ions, weak stimulation normally activates only the AMPA

receptors, resulting in a slight depolarization of the postsynaptic neuron.

When glutamate binds to the NMDA receptor at slightly depolarized or resting membrane

voltages, very few ions flow through the channel. This low conductance occurs because the pore of

the channel is blocked by Mg2+ ions, which prevent other ions from passing freely through

the channel.

Given a stimulus of sufficient strength or frequency, AMPA receptors can Depolarize the

membrane sufficiently to expel the Mg2+ from the NMDA Channel. The NMDA channel now actively responds to glutamate, Admitting not only Na+, but large amounts of Ca2+ as well. The Calcium

acts as an important second messenger, activating several intracellular signaling cascades.

Some of this calcium binds to calmodulin, and this complex in turn activates several protein kinases, including calcium/calmodulin dependant proteinkinase, or CAM kinase. CAM kinase affects AMPA receptors in two ways. First, it phosphorylates

AMPA receptors already present in the dendritic spine membrane, therby increasing their

conductance to sodium ions.

CAMKII also promotes movement of AMPA receptors from intracellular stores into the membrane,

making more receptors available to stimulate the spine. In addition to these postsynaptic effects,

Ca2+ may also facilitate the release of transmitter from the presynaptic axon terminal via retrograde

signals, such as nitric oxide (NO).

As a result of the increase in the number of AMPA receptors, the response to a stimulus of a given strength will be stronger than it was before the NMDA receptors were activated. In this regard, the synapse is said to be “enhanced” and this

physiological change is thought to be one of the mechanisms underlying the expression of long-

term potentiation, or LTP.

LITERATURE CITEDCain, et.al. 2006. NMDA and AMPA receptors.Available from: http://www.sumanasinc.com/webcontent/anisamples/neurobiology/receptors.html Accessed 3/27/2007

Krusz, J.C., Robert, T., 2005 November 5. Namenda™ for Chronic Headache and Migraine Prevention. Available from: http://www.helpforheadaches.com/articles/memantine.htm. Accessed 3/27/2007

Parsons, C.G., Danysz, W., and Quack, G. 1999. Memantine is a clinically well tolerated N-methyl-D-aspartate (NMDA) receptor antagonist- a review of preclinical data. Neuropharmacology. 38: 735-767.

Thisse, C., Thisse, B. 2008. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nature Protocols. 3 (1): 59-69.

Li, X., Xiong, J.W., Shelley, C.S., Park, H., Arnaout, M.A. 2006. The transcription factor ZBP-89 controls generation of the hematopoietic lineage in zebrafish and mouse embryonic stem cells. Development. Sep;133(18):3641-50. Epub 2006 Aug 16.

ACKNOWLEDGEMENTS

I would like to thank Dr. Wendy Boehmler for sharing her expertise and providing valuable guidance through the progression of my Senior

Thesis research project. I would also like to thank The Pennsylvania State University Cellular & Molecular Biology Department for

graciously providing the VE-cadherin probe for in situ hybridization.

Experimental GoalsTo stimulate a migraine in the zebrafish embryos by LTP with glutamate (neurotransmitter)

To have groups of glutamate only, memantine only and glutamate pre-treated with memantine in addition to one control group.

Compare experimental vs. control groups and note differences in blood vessel diameters.

HYPOTHESIS

H0: There is no difference in blood vessel diameter when comparing memantine pre-treated embryos and non pre-treated embryos.

INTRODUCTION

Curtis Gallagher • Department of Biological Sciences • York College of Pennsylvania

• Magnesium block expelled at –50mV

• Memantine stays in the NMDA channel longer (-20mV)

• Decreased chance and/or frequency of Nitric Oxide release

Proper in situ hybridization staining of zebrafish embryo blood vessels probing for flk1 (one of the VEGF receptors) by Li (2006).

No specific in situ hybridization staining of zebrafish embryo blood vessels when probing for VE-cadherin. The staining is an “all or nothing” response and the slightly darker area running down the tail is likely non-specific staining.

CONCLUSIONSNo specific blood vessel staining was achieved within situ hybridization likely due to RNAse contamination. Therefore the hypothesis cannot be evaluated at this time.

RNAse could have contaminated the washes, the equipment, or even the embryo itself, degrading the RNA probe needed to visualize the vessels for quantification.

FUTURE STUDIESThe benefit of memantine as a prophylactic treatment in migraine was unable to be determined in this study. The theory could be re-evaluated, possibly utilizing another staining technique such as microinjection of a dye in order to visualize any vessel diameter differences.

Embryos dechorionated @ 20hpf using Pronase™ enzymeEmbryos dechorionated @ 20hpf using Pronase™ enzyme

Adult zebrafish bred & embryos collected

Embryos placed in groups & drug(s) applied @ 22hpf

Control 6.8 μMGlutamate

6.8 μM Glut& 3mM mem

6.8 μM Glut& 10mM mem

Glut = Glutamatemem = memantine HCl

6.8 μM Glut& 30mM mem

3mMmemantine

10mMmemantine

30mMmemantine

Embryos fixed in 4% PFA @ 24hpf then placed in MeOH

Initial measurement tool too ambiguous to accurately measure blood vessels

Alternative measurement method found, in situ hybridization to be performed

VE-cadherin DNA obtained (blood vessel specific)

VE-cadherin DNA transformed into E. coli bacteria

DNA isolated & purified using Qiagen™ mini-prep

RNA probe synthesized using template DNA and T3 promoter (transcription)

In situ hybridization procedure started

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